1. Field of the Invention
The present invention relates to the field of evaluation of agonists or antagonists of wild-type receptors, i.e., cellular receptors that have not been chemically or genetically modified for assay purposes.
2. Related Art
Presented below is background information on certain aspects of the present invention as they may relate to technical features referred to in the detailed description, but not necessarily described in detail. That is, individual parts or methods used in the present invention may be described in greater detail in the materials discussed below, which materials may provide further guidance to those skilled in the art for making or using certain aspects of the present invention as claimed. The discussion below should not be construed as an admission as to the relevance of the information to any claims herein or the prior art effect of the material described. G protein-coupled receptors (GPCRs) form one of the largest families of integral membrane receptors. GPCRs transduce information provided by extracellular stimuli into intracellular second messengers via their coupling to heterotrimeric G proteins and the subsequent regulation of a variety of effector systems. Therapeutic agents often target GPCRs because of their capability to bind ligands, hormones, and drugs with high specificity. Agonist activation of GPCRs also initiates processes that desensitize GPCR responsiveness and their internalization.
Common to most GPCRs is the cyclic process of signaling, desensitization, internalization, resensitization, and recycling to the plasma membrane. This cycle prevents cells from undergoing excessive receptor stimulation or periods of prolonged inactivity. Mechanisms for desensitization of GPCRs include receptor phosphorylation and subsequent endocytosis, which removes the receptor-ligand complex from the cell surface. As a result of this desensitization process, a common limitation of GPCR-targeted compositions is target organism tolerance or resistance, as receptor desensitization can mute their effectiveness.
U.S. Pat. No. 7,235,374 describes mutant GPCRs incorporating serines and/or threonines in the C-terminal region of the GPCR and using β-galactosidase fragments for detection. See also, as illustrative of activity in the field, Hammer, et al. 2007, FASEB J. 21, 3827-34; Molinari, et al. 2008, Biochem. J. 409, 251-61; Hamdan, et al. 2007, J. Biol. Chem. 282, 29089-100; Garippa, et al. 2006 Methods Enzymol. 414, 99-120; and Yan, et al. 2002 J. Biomol. Screen. 7, 451-9.